Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex—SecYEG in the inner membrane—is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

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Further information is available in our Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguide We realize that it is difficult to revise to a specific deadline.In the interest of protecting the conceptual advance provided by the work, we recommend a revision within 3 months (31st Oct 2023).Please discuss the revision progress ahead of this time with the editor if you require more time to complete the revisions.Use the link below to submit your revision: - -----------------------------------------------Referee #1: The Sec translocon is the major route for protein export across the cytoplasmic membrane of prokaryotes (and essential the same translocon is responsible for protein entry into the eukaryotic endoplasmic reticulum).Thus, detailed mechanistic studies of the operation of the Sec translocon are of great impotance for cellular biology.In bacteria protein transport through the Sec translocon is normally post-translational and powered by the ATP-driven `motor' SecA.In this study the authors have used cutting edge single molecule FRET measurements to characterize the kinetics of opening of the Sec translocon and how these are modulated by SecA during different stages in the cycle of ATP hydrolysis and substrate engagement.From these measurements the authors provide a convincing case that the role of ATP hydrolysis is to enable SecA to bias the opening of the Sec channel to allow diffusive movement of the substrate protein.This contrasts with the majority view that the substate protein is mechanically pushed in a power stroke mechanism.This conclusion is a paradigm shift in our understanding of Sec mechanism.The work will be of great significance for the protein transport field and more generally for the understanding of biological machines.The work is extremely well presented and argued, given the complexity of the methods.The technical nature of the work is outstanding and thorough.My comments on the manuscript are mainly presentational.
This study relies on the assignment of the two FRET states that are identified and analyzed as corresponding to the physiological open and closed states of the translocon.The evidence for this assignment is given as (Allen et al, 2016).Whilst I agree that the assignment is highly plausible and that the data in Allen et al. is consistent with this interpretation, that paper does not seem to report FRET measurements from states unambiguously trapped in the two states.For this reason, I would recommend rather than just citing Allen et al. that the authors summarize the key reasons why that paper supports the assigned states.
It is absolutely vital to the interpretation of the data that the reader knows that the SecYEG complexes have been reconstituted into proteoliposomes.I didn't initially pick up that this was what the authors had done even though it is mentioned where the main text talks about labelling and shown in Fig. 2A.It might be worthwhile to state in the figure legends that the samples being analysed are proteoliposomes.Part of my confusion was that the methods describe isolation of SecYEG in detergent but as far as I can see do not describe reconstitution into proteoliposomes.
If the Sec translocon is 20% open without partners (apo state) as the authors observe how does the cell maintain the membrane seal?The authors should also note and comment on previous electrophysiology data (Saparov et al. (2007) Mol Cell 26: 501-509) that indicated that the apo state does not gate open and that the PrlA4 variant shows opening for extended periods of time.
Pg 2. `The complex of the two (SecYEG:A) is necessary and sufficient for the translocation of unfolded polypeptides across the inner membrane (Brundage et al, 1990;Arkowitz et al, 1993).'It would be advisable to qualify this as `in vitro' as there are studies that claim that PMF is essential for transport in vivo.
Pg 4. `...there is less than 1 nm available for 2HF movement, not enough for the proposed power stroke.'It is not obvious to me why a movement of this scale would not be enough for a powerstroke.Either give the more detailed rationale or qualify as `unlikely to be enough'.
Pg 11. `Thus, the rate limiting step is a conformational change that involves the SecYEG channel and adjacent LG and is associated with the conversion of SecYEG:A:ADPPH to a state from which ADP is readily released, most likely the equilibrium SecYEG:A:ADPL state.'This needs to be rewritten for clarity because I'm not sure I have followed what is being argued here.I can see that the rate limiting step is as described in the second half of the sentence but what is the link to SecYEG conformational change?If this is that the two states have different SecYEG opening regimes then this connection should be made.Fig. 2B.Even if conventional in the field, the presentation here is difficult to understand as it looks as though the burst are higher and lower than a baseline whereas the two halves of the graph refer to bursts at two different emission wavelengths (I assume).So either label the two halves on the y-axis as being associated with different wavelengths and/or have the acceptor emission trace as a replica panel going from 0 to 1 rather than from 1 to 0. Similarly, in 2C it needs to be clearer in the legend and figure labelling that the y-axis scale is plotting the running average/modelled FRET states and that the vertical lines are just time stamps for photons and not related to the y-axis quantities.

Fig 5d.
It would be preferable to label in this figure which is the variant data and which the wild type rather than relying on carrying over colours from other panels.What are the grey dotted lines?Are they necessary?Should they be arrows?Fig 5e.What do the comments written on the panel mean?Topen is presumably from the data shown but is the efficiency of translocation is from elsewhere and so can't be calculated from this graph?This should be made clear.

Fig 6a.
It should be made clear that the y-axis scale is proportional and assigned from quantitative calculations (rather than being illustrative) but that the scale is relative not absolute.Maybe have numbers on the axis and label these as relative delta G?Although the calculation is given in the SI methods the nature of the calculation in the figure legend is too minimal.What is the nature of the `data' that are going into the calculation to produce state thermodynamics?It was not clear to me what had been done even from the methods.

Referee #2:
The precision of protein transport by the Sec translocon is vital for all aspects of life.However, the underlying molecular mechanism remain elusive.In this manuscript, Crossley et al. investigated the fundamental principle of protein translocation using single molecule FRET and biochemical reconstitution.The results depicted a comprehensive framework of the translocon dynamics during the ATP hydrolysis cycle of SecA, demonstrating protein translocation occurs mostly likely through the Brownian ratchet model instead of the power stroke model as proposed by previous studies based on ensemble biochemical measurements.Therefore, the findings reported by Crossley et al. resolve a long-standing debate about the Sec translocon and provide novel insights into our understanding of protein translocation.The results are very exciting and will become a 'must-read' paper for people interested in single molecule biophysics.The manuscript is well-written with self-explained figures.I am highly supportive in the publication of the manuscript if a few minor issues could be clarified as detailed below.
1.One potential issue is that all smFRET measurements were performed in proteoliposomes in which 50% of SecY will not be available for engaging SecA or substrate.To address this problem, the authors carried out extensive simulations to determine the "corrected rates".However, these data were not included in the paper.As such, it is challenging to understand the processing of the smFRET data.It would be very useful if the authors could provide these simulations and also a more detailed procedure in the analysis of their smFRET data.
2. The other issue of using proteoliposomes is the copy number of the Sec translocon.For single molecule imaging experiments, it is critical to toss out oligomers from monomers.However, I am not able to figure out how the authors know that the proteoliposomes analyzed in this study only contain monomers.
3. Since the authors have determined the rate constants in the conformational transitions of the Sec translocon, it should be possible to calculate the energy of each state and include those values in the free energy profiles.
Referee #3: Crossley et al. reported the opening dynamics of the SecYEG channel in different translocation states using smFRET.SecYEG is one of the most conserved and ubiquitous channels (known as Sec61 in eukaryotes) responsible for translocating secretory proteins across lipid membranes.In bacteria, the channel is coupled with the ribosome for co-translational translocation of nascent peptide chains, and with SecA for post-translational translocation of peptide chains.SecA is a highly conserved ATPase that provides energy for protein translocation.The translocation mechanism of SecA with SecYEG has been studied for decades, with two major working models currently under debate: the Brownian ratchet model and the power-stroke model.The authors' group has published a series of papers supporting the Brownian ratchet model, and they have employed smFRET with similar experimental settings in their previous work (Allen et al., elife 2016).This manuscript presents smFRET data from faster recording and an improved fitting model.With these improvements, the authors could better define the open and close frequencies at the lateral gate of the channel in different nucleotide and SecA binding states.The different timescales observed between the dynamics of the SecY gate and SecA hydrolysis led the authors to conclude that SecA modulates SecYEG conformation to facilitate protein translocation.While the refined measurements of SecYEG dynamics is informative, the current data is too preliminary to draw the conclusion.
Major points: 1.The estimated timescale of ATP hydrolysis by SecA was based on other biochemical assays.As smFRET is super sensitive to conformational changes, the dynamics of SecA in the similar smFRET settings should be measured as well to justify the conclusion.The authors could label different domains of SecA, measure the internal dynamics, and assess the distance between SecA and SecYEG in different nucleotide and translocation states.2. The PH state is puzzling.The use of different nucleotides to define the ATP hydrolysis states should be done with caution.For example, in the ADP state, SecA may rapidly alternate between being on and off from the channel.It is unclear whether the reported gate dynamics reflect the average behavior (once again, the dynamics of SecA in this state should be measured).The exact states mimicked by ATP analogs, such as ATPγS, AMPPNP, ADP.BeFx, and ADP.AlFx, have been interpreted differently in various ATPase systems.Notably, the AMPPNP data was reported in previous work (Allen et al., elife 2016), but not here.Any specific reasons?This reviewer suggests including more supporting data to clearly define each state, beyond the apparent gate opening and closing frequencies.Additional data would strengthen the study's conclusions and provide a more comprehensive understanding of the PH state (if exists).3. The discussion on the rate limiting step in Page11 lines 3-8 is unclear.Please elaborate how such conclusion is drawn.4. The authors suggest that the leakage of the Prl4 mutant may be explained by the longer open state.However, the overall translocation rates are supposed to reflect the average of open and closed states.If we consider the ratio of open and closed states, the wild-type (wt) actually appears to be even better than Prl4 (compare figs 4b and 5e). 5.In the last sentence of the abstract, the authors seem to infer that the channel is static according to previous structural studies.However, this is not true.It is well known from structural and biochemical studies that the SecYEG channel is dynamic and can open up to accommodate substrates of different sizes.SecA binding also contributes to opening up and tightening the channel.In the main text, while providing a detailed description of the channel dynamics, the authors should refrain from overemphasizing the importance of their results by suggesting that the SecA-mediated dynamic nature of the channel was not known from previous structural data. 1

Referee #1:
The Sec translocon is the major route for protein export across the cytoplasmic membrane of prokaryotes (and essential the same translocon is responsible for protein entry into the eukaryotic endoplasmic reticulum).Thus, detailed mechanistic studies of the operation of the Sec translocon are of great impotance for cellular biology.
In bacteria protein transport through the Sec translocon is normally post-translational and powered by the ATP-driven 'motor' SecA.In this study the authors have used cutting edge single molecule FRET measurements to characterize the kinetics of opening of the Sec translocon and how these are modulated by SecA during different stages in the cycle of ATP hydrolysis and substrate engagement.From these measurements the authors provide a convincing case that the role of ATP hydrolysis is to enable SecA to bias the opening of the Sec channel to allow diffusive movement of the substrate protein.This contrasts with the majority view that the substate protein is mechanically pushed in a power stroke mechanism.This conclusion is a paradigm shift in our understanding of Sec mechanism.The work will be of great significance for the protein transport field and more generally for the understanding of biological machines.
The work is extremely well presented and argued, given the complexity of the methods.The technical nature of the work is outstanding and thorough.My comments on the manuscript are mainly presentational.
This study relies on the assignment of the two FRET states that are identified and analyzed as corresponding to the physiological open and closed states of the translocon.The evidence for this assignment is given as (Allen et al, 2016).Whilst I agree that the assignment is highly plausible and that the data in Allen et al. is consistent with this interpretation, that paper does not seem to report FRET measurements from states unambiguously trapped in the two states.For this reason, I would recommend rather than just citing Allen et al. that the authors summarize the key reasons why that paper supports the assigned states.

R1-A1:
We have now expanded the opening of the results section to include a summary of the data in (Allen et al, 2016) that provides evidence for the FRET mutant SecYA103C-V353CEG reporting on the opening and closure of the SecY lateral gate.
It is absolutely vital to the interpretation of the data that the reader knows that the SecYEG complexes have been reconstituted into proteoliposomes.I didn't initially pick up that this was what the authors had done even though it is mentioned where the main text talks about labelling and shown in Fig. 2A.It might be worthwhile to state in the figure legends that the samples being analysed are proteoliposomes.Part of my confusion was that the methods describe isolation of SecYEG in detergent but as far as I can see do not describe reconstitution into proteoliposomes.

R1-A2:
We have now made reference to proteoliposomes in the Results section and figure legends.We have also split the proteoliposomes reconstitution protocol into its own section in the Methods.If the Sec translocon is 20% open without partners (apo state) as the authors observe how does the cell maintain the membrane seal?The authors should also note and comment on previous electrophysiology data (Saparov et al. (2007) Mol Cell 26: 501-509) that indicated that the apo state does not gate open and that the PrlA4 variant shows opening for extended periods of time.
R1-A3: Referee #1 brings up a great point here and highlights the importance of consolidation of our data showing spontaneous opening of channel with previous electrophysiology data showing that apo wild-type SecY is impermeable to ions and water (Saparov et al. 2007).We have added a paragraph to the discussion section explaining how we believe these two findings can be reconciled.Pg 2. `The complex of the two (SecYEG:A) is necessary and sufficient for the translocation of unfolded polypeptides across the inner membrane (Brundage et al, 1990;Arkowitz et al, 1993).'It would be advisable to qualify this as `in vitro' as there are studies that claim that PMF is essential for transport in vivo.
R1-A4: We thank Referee #1 for pointing out this important distinction which we have now added to the manuscript.Pg 4. `...there is less than 1 nm available for 2HF movement, not enough for the proposed power stroke.'It is not obvious to me why a movement of this scale would not be enough for a powerstroke.Either give the more detailed rationale or qualify as `unlikely to be enough'.

R1-A5:
We have amended the text to state 'unlikely to be enough for the power stroke as proposed in the above papers.'and we agree with Referee #1 that this is appropriate wording given the statement's subjective nature.We also expanded the end of the paragraph to clarify the reasoning behind our point.
Pg 11. `Thus, the rate limiting step is a conformational change that involves the SecYEG channel and adjacent LG and is associated with the conversion of SecYEG:A:ADPPH to a state from which ADP is readily released, most likely the equilibrium SecYEG:A:ADPL state.'This needs to be rewritten for clarity because I'm not sure I have followed what is being argued here.I can see that the rate limiting step is as described in the second half of the sentence but what is the link to SecYEG conformational change?If this is that the two states have different SecYEG opening regimes then this connection should be made.R1-A6: We agree with Referee #1 that the terminology used here was confusing.We have significantly expanded this section and used terminology which we think makes the text easier to follow.Fig. 2B.Even if conventional in the field, the presentation here is difficult to understand as it looks as though the burst are higher and lower than a baseline whereas the two halves of the graph refer to bursts at two different emission wavelengths (I assume).So either label the two halves on the y-axis as being associated with different wavelengths and/or have the acceptor emission trace as a replica panel going from 0 to 1 rather than from 1 to 0.
Similarly, in 2C it needs to be clearer in the legend and figure labelling that the y-axis scale is plotting the running average/modelled FRET states and that the vertical lines are just time stamps for photons and not related to the y-axis quantities.R1-A7: We thank the reviewer for these suggestions which we have implemented, making the figures easier to understand.The Figures/Legends are amended accordingly.It should be made clear that the y-axis scale is proportional and assigned from quantitative calculations (rather than being illustrative) but that the scale is relative not absolute.Maybe have numbers on the axis and label these as relative delta G?Although the calculation is given in the SI methods the nature of the calculation in the figure legend is too minimal.What is the nature of the `data' that are going into the calculation to produce state thermodynamics?It was not clear to me what had been done even from the methods.R1-A9: We have amended Fig 6A to include the changes Referee #1 has suggested.We also changed the introduction to the Appendix section 'Calculation of simplified potential energy surface' to make it clear that the data used was the transition rates.

Referee #2:
The precision of protein transport by the Sec translocon is vital for all aspects of life.However, the underlying molecular mechanism remain elusive.In this manuscript, Crossley et al. investigated the fundamental principle of protein translocation using single molecule FRET and biochemical reconstitution.The results depicted a comprehensive framework of the translocon dynamics during the ATP hydrolysis cycle of SecA, demonstrating protein translocation occurs mostly likely through the Brownian ratchet model instead of the power stroke model as proposed by previous studies based on ensemble biochemical measurements.Therefore, the findings reported by Crossley et al. resolve a long-standing debate about the Sec translocon and provide novel insights into our understanding of protein translocation.The results are very exciting and will become a 'must-read' paper for people interested in single molecule biophysics.The manuscript is well-written with self-explained figures.I am highly supportive in the publication of the manuscript if a few minor issues could be clarified as detailed below.
1.One potential issue is that all smFRET measurements were performed in proteoliposomes in which 50% of SecY will not be available for engaging SecA or substrate.To address this problem, the authors carried out extensive simulations to determine the "corrected rates".However, these data were not included in the paper.As such, it is challenging to understand the processing of the smFRET data.It would be very useful if the authors could provide these simulations and also a more detailed procedure in the analysis of their smFRET data.R2-A1: We apologise that this was not clear.We have now added a figure outlining our 'Apo correction' process (Appendix Fig S4) and clarified text in the Appendix section 'Correction for the presence of apo SecYEG', which we refer to in the main text.
2. The other issue of using proteoliposomes is the copy number of the Sec translocon.For single molecule imaging experiments, it is critical to toss out oligomers from monomers.However, I am not able to figure out how the authors know that the proteoliposomes analyzed in this study only contain monomers.R2-A2: We have updated our renamed Materials and Methods section 'SecYEG Proteoliposome Preparation' to better clarify how we prepared proteoliposomes without more than 1 copy of SecYEG.

Referee #3:
Crossley et al. reported the opening dynamics of the SecYEG channel in different translocation states using smFRET.SecYEG is one of the most conserved and ubiquitous channels (known as Sec61 in eukaryotes) responsible for translocating secretory proteins across lipid membranes.In bacteria, the channel is coupled with the ribosome for co-translational translocation of nascent peptide chains, and with SecA for post-translational translocation of peptide chains.SecA is a highly conserved ATPase that provides energy for protein translocation.The translocation mechanism of SecA with SecYEG has been studied for decades, with two major working models currently under debate: the Brownian ratchet model and the power-stroke model.The authors' group has published a series of papers supporting the Brownian ratchet model, and they have employed smFRET with similar experimental settings in their previous work (Allen et al., elife 2016).This manuscript presents smFRET data from faster recording and an improved fitting model.With these improvements, the authors could better define the open and close frequencies at the lateral gate of the channel in different nucleotide and SecA binding states.The different timescales observed between the dynamics of the SecY gate and SecA hydrolysis led the authors to conclude that SecA modulates SecYEG conformation to facilitate protein translocation.While the refined measurements of SecYEG dynamics is informative, the current data is too preliminary to draw the conclusion.
Major points: 1.The estimated timescale of ATP hydrolysis by SecA was based on other biochemical assays.As smFRET is super sensitive to conformational changes, the dynamics of SecA in the similar smFRET settings should be measured as well to justify the conclusion.The authors could label different domains of SecA, measure the internal dynamics, and assess the distance between SecA and SecYEG in different nucleotide and translocation states.
R3-A1: We agree with the referee that the dynamic coupling in the Sec translocon needs to be investigated further to all the other factors associated with translocation (SecA and many others) ideally simultaneously with those in SecYEG.However, doing this will be technically very challenging and will form the basis of a whole series of experiments; enough to from another manuscript.This will obviously take some time, beyond the scope of this study, but will hopefully form the basis of our next submission!We have written a section of text summarising the technical challenges in the Discussion section 'Fast protein channel and LG dynamics are integral to the mechanism of translocation' second paragraph.
Overall, however, we do not believe this omission undermines the important conclusions of the manuscript.Previous research by the Rapoport Lab used total internal reflection fluorescence microscopy to measure smFRET on SecA and recover the rates of conformational change during protein transport similar to the way Referee #3 suggests (Catipovic et al. 2019).Importantly, the results showed that the rates matched well to the ensemble ATP hydrolysis rates measured by their group and are similar to the values which we present here, suggesting that the method of measurement has little quantitative effect on the determined values.
Furthermore, we would note that the important new conclusions of our paper do not rest on the exact rate constants within the ATPase cycle.The evidence is very strong for the mismatched timescales between the conformational dynamics of SecYEG and SecA, and for the assignment of different conformational equilibria to specific nucleotide states.It is upon these robust comparisons that we make our conclusions.
2. The PH state is puzzling.The use of different nucleotides to define the ATP hydrolysis states should be done with caution.For example, in the ADP state, SecA may rapidly alternate between being on and off from the channel.It is unclear whether the reported gate dynamics reflect the average behavior (once again, the dynamics of SecA in this state should be measured).The exact states mimicked by ATP analogs, such as ATPγS, AMPPNP, ADP.BeFx, and ADP.AlFx, have been interpreted differently in various ATPase systems.Notably, the AMPPNP data was reported in previous work (Allen et al., elife 2016), but not here.Any specific reasons?This reviewer suggests including more supporting data to clearly define each state, beyond the apparent gate opening and closing frequencies.Additional data would strengthen the study's conclusions and provide a more comprehensive understanding of the PH state (if exists).

R3-A2:
The reviewer is absolutely correct that the exact interpretation of different nucleotide analogues should be treated with caution.Concerning the first part of the question, i.e. the PH statewe consider it highly unlikely that SecA dissociation can explain the SecYEG:A:ATP state.Firstly, the PH state has a dynamic signature in the 2D graph that is very different from both 'apo' and ADP states.More generally, if SecA were dissociating and rebinding on a timescale fast enough to contribute to a dynamic average (i.e.ms timescale), it would presumably also be doing this with ADP present.If so, this would already be reflected in the SecYEG:A:ADP data, which has clearly different dynamics.Thus, we only invoke the PH state because it seems impossible to explain the SecYEG:A:ATP data in any other way.We have rewritten the relevant section to make this argument clearer.
With regards to the initial choice of ATPγS over AMPPNP, the reason was two-fold.1) Research by Fak et al. which showed that AMP-PNP may not serve as faithful mimic of ATP in its interactions with SecA due to its affinity to SecA being at least 100x weaker than ATPγS (Fak et al. 2004).2) ATPγS has been used in numerous studies on SecA-SecY interactions as a non-hydrolysable analogue and we wanted to be able to compare our data with these other reports, most notably smFRET by Catipovic et al. 2019 (important also for response to point 1 above); Bauer et al. 2014;Catipovic and Rapoport 2020.However, we have now also collected data for AMP-PNP (five independent hour long data acquisitions, measured in the same way as all other conditions).The data for ATPγS and AMP-PNP match well (now shown in new Appendix Figs.S13 and S14 and Appendix Table 1) giving us further confidence that the dynamic ensemble we measured matches the pre-hydrolysis state in the SecYEG:A complex and allows us to compare our present conclusions with our previous TIRF data as well as the more recent work on SecA by others.
3. The discussion on the rate limiting step in Page11 lines 3-8 is unclear.Please elaborate how such conclusion is drawn.R3-A3: We apologise this was not clear.We have now significantly expanded this section to better explain the conclusion to our findings and think this paragraph is now much better.Thank you for pointing this out.
4. The authors suggest that the leakage of the Prl4 mutant may be explained by the longer open state.However, the overall translocation rates are supposed to reflect the average of open and closed states.If we consider the ratio of open and closed states, the wild-type (wt) actually appears to be even better than Prl4 (compare figs 4b and 5e).
R3-A4: At first, we also expected to see the PrlA4 mutant spending a greater proportion of its time in the open state, to account for the increased transport rate.However, as the reviewer points out, this is not what the data show.Therefore, we must conclude that it is the dwell time in the open state, rather than the ratio of open and closed states, that determines overall transport rate.
Our current working hypothesis is that a successful 'transport event'i.e.passage of a difficult stretch of pre-protein across the membranerequires the channel to stay open for a relatively long time (say 4 ms).Therefore, transport time is dictated by the frequency of long-duration opening events, not just the average channel position.This would explain why PrlA4 transports much faster despite spending the same (or more) time in the closed state.We suggest that the longer dwell time in the closed state is inconsequential.
We think this is an important observation, and as the referee points out, is perhaps counterintuitive.We have amended the section where this is discussed to make it clearer.
5. In the last sentence of the abstract, the authors seem to infer that the channel is static according to previous structural studies.However, this is not true.It is well known from structural and biochemical studies that the SecYEG channel is dynamic and can open up to accommodate substrates of different sizes.SecA binding also contributes to opening up and tightening the channel.In the main text, while providing a detailed description of the channel dynamics, the authors should refrain from over-emphasizing the importance of their results by suggesting that the SecA-mediated dynamic nature of the channel was not known from previous structural data.
R3-A5: We absolutely did not intend to imply that previous studies show a static SecYEG channel.Rather, we were attempting to highlight the importance of rapid dynamics and energy landscape steering for the mechanism of translocation.These ms-timescale dynamics are the major new insight of our manuscript, which have been made possible by recent advances in smFRET methodology and analysis.
We have amended and extended the final section of the abstract to make this clearer, and added better descriptions of the dynamic equilibrium and how it differs from previous results to the main text.
Together, we believe these changes improve the manuscript to better reflect the novelty of our findings.We hope that it is now clear that the manuscript goes beyond the known properties of SecA to alter the conformation of SecY.Rather, the dynamics of the Sec machinery are manifested through multiple timescales orders of magnitude apart, modulated allosterically within SecYEG via energy landscape steering.We believe that this concept of dynamic allostery will not only be essential to understand how protein translocation occurs in the Sec machinery, and other systems, but have wider implications in understanding other complex molecular machines through cutting edge single-molecule fluorescence techniques.

28th Sep 2023 1st Revision -Editorial Decision
Dear Ian, I have now received comments from all three referees on the revised version of your manuscript.I have included these reports at the bottom of this email.As you will see, referees 1 and 2 are satisfied by the changes that you have made.Unfortunately, referee 3 remains unconvinced that your experimental design is able accurately to test the hypotheses which you present.This report is clearly written and well argued.The technical nature of referee 3's concerns makes it impossible for me to accept the manuscript for publication in its current form.However, to be completely fair to you and your co-authors, I would like to offer you another opportunity to respond to the points raised and find common ground.Please let me know if you judge that another round of experiments could help, as this possibility is also still open.

Referee #2:
In this manuscript, Crossley et al. provided novel insights into the molecular mechanism of the protein translocon using state-ofart single molecule approaches.The authors have well addressed my previous concerns.I am delighted to support the publication of this study at EMBO J.

Referee #3:
The revised manuscript has improved clarity in writing.However, a major concern still persists regarding the interpretation of the dynamics of SecY in various nucleotide binding states, particularly with the inclusion of the AMPPNP data in the revision.As previously pointed out in the comments, it is essential to exercise caution when defining ATP hydrolysis states using different nucleotide analogs.In this case, the FRET data indicates that, when combined with SecA, the SecY channel is mostly in the closed state with ADP, which is consistent with other biochemical and structural data.However, the FRET data obtained with different ATP analogs are not consistent with each other.1. ATPγS is typically considered a slowly hydrolysable ATP analog, rather than strictly a non-hydrolysable ATP, as stated in the manuscript.In the FRET experiment setting (with recordings lasting ~ 1 hour), it becomes challenging to estimate the extent to which ATPγS has been converted to ADP.For instance, ATPγS has been employed in many AAA-ATPase systems to capture subunits in various states, including ATP, ADP, and apo states, for biochemical and structural studies.Thus, it is not convincing to treat the ATPγS data as the pre-hydrolysis state with the current experimental settings.reported that AMPPNP exhibited a much lower affinity for SecA compared to ATPγS, and even lower than ADP.However, the FRET data reported in this study indicate that AMPPNP and ATPγS resulted in similar SecY dynamics, which is difficult to reconcile.More notably, the AMPPNP data in this manuscript appear to contradict the findings from an earlier paper by the same group, Allen WJ et al. eLife 2016.In that earlier work, AMPPNP was regarded as a non-hydrolysable ATP analog, and its FRET data showed significant stimulation of SecY channel opening, to an extent even greater than ATP binding (Fig. 5 of the paper).Considering these conflicting data and interpretations, it is quite challenging to persuade this reviewer of the conclusions drawn regarding the dynamics of the channel during protein translocation solely based on the FRET data and subsequent analyses.Concerning the explanation that Τopen of Prl4 dictates the translocation process, do the authors think that a single opening event (e.g., within 4 ms) could allow the passage of an entire protein strand?If not, when a protein peptide is present in the channel without signal sequence peptides (SS), will the FRET signal indicate an open state or a closed state?In other words, do the FRET experiments in this report distinguish between the opening of the lateral gate induced by SS (at the initialization stage, fig.4a), SS and a translocating peptide inside the channel (at the early translocation stage), and a peptide alone in the channel (at the middle and later translocation stages)?The authors do not seem to have considered this difference.However, in the reviewer's opinion, it is crucial to define the relationship between these various "open" states and the FRET signal before proceeding with further analysis.Presumably, an idle channel has a closed lateral gate (LG), an active channel with SS and a translocating peptide has an open LG.An active channel with only a translocating peptide has an LG opening somewhere between these two states.Neglecting to address this issue could potentially affect the interpretation of the Τopen/Τclosed plots for the ATP+proSpy sample.
1 Dear Dr Teale, Thank you for sending us the referees' comments on our revised manuscript.We were pleased to see that referees 1 and 2 recommend publication.We are grateful for the opportunity to respond reviewer 3; because, fundamentally, their second round of objections do not stand up to technical scrutiny.Rather, the referee appears to have preconceived opinions about how ATPases ought to work, which are incompatible with our results.As such, more datahowever compellingis unlikely to make any difference to their viewpoint.
The main point of contention is the assignment of the state observed in the presence of ATPγS and AMPPNP (the latter performed at the referee's suggestion).These are slowly-and nonhydrolysing analogues of ATP (respectively), and are widely used to capture pre-hydrolysis conformations of ATPases.While both differ slightly from ATP, they clearly represent prehydrolysis conformations because the terminal phosphate (or thiophosphate) is covalently bonded to the ADP moiety, and not prone to hydrolysis.Furthermore, the results we present with both of these ligands agree with one another, confirming that the pre-hydrolysis conformation we observe is not just a quirk of one particular analogue.
The referee objects to this assignment, but does not provide any alternative interpretation of the data.Instead, they make spurious arguments against each analogue individually: They suggest ATPγS might be being hydrolysed over the course of the experiment to the extent that it would lead us to misinterpret our data.This is not plausible in a single molecule setupeven in the case of ATP, let alone a slowly hydrolysing analogue.Our experiment contains 1 µM SecA, 1 mM ATP, and negligible (30 pM) SecYEG.As the basal turnover rate of SecA is 0.56 min -1 (Robson et al. 2009), simple calculations suggest ~34 µM ATP will be turned over in an hour (60 min * 0.56 min -1 * 1 µM SecA)i.e. 3.4% of the total ATP.For ATPγS this figure will be much lower.Consistent with this, there is no change in FRET behaviour over the course of data collection (see attached figure).
The second argument is that ATPγS is often used:"to capture […] ADP, and apo states".This defies reason, and no supporting literature is provided.To capture an ADP-bound state, why add ATPγS instead of ADP?And to capture the apo state, why add nucleotide at all?We presume that this statement might be based on crystal structures, wherein ATPγS may not be captured in a bound state (yielding apo), or where the high protein concentrations and long time scales (weeks) of crystallisation may enable hydrolysis to occur.This would definitely NOT be the same for 1 h acquisition in solution under the single molecule conditions we employ.
For AMPPNP, which very clearly cannot be hydrolysed, the referee instead claims that the lower affinity of SecA for AMPPNP means it should behave differently to ATPγS.But we are working at saturating nucleotide concentrations (1 mM, where the affinity is 50 µM according to Fak et al. 2004), so the affinity is irrelevant and therefore the data should, and does, look the same.

Dr William Teale Scientific Editor
The EMBO Journal The assertion that the data contradict our previous findings seems to be based on direct comparison of two data sets without consideration for the different ways they were obtained and interpreted.In the previous paper (Allen et al., 2016) we were unable to follow fast dynamics and assumed (incorrectly) that changes in SecY occur on the same time scale as those of the SecA ATPase.Based on this assumption and available structural data we interpreted the FRET patterns in terms of three static structural states.However, using better time resolution and the recently developed advanced data processing methodology we show that the data can be explained by just two states with rapid exchange between them.The fact that results of the two studies qualitatively agree is the best that can be expected, given that the mismatch between the number of states and their dynamic nature make direct quantitative comparison impossible.
As none of the above criticisms pass scrutiny, the sensible conclusion is that AMPPNP and ATPγS do in fact accurately represent the ATP-bound (pre-hydrolysis) stateas is widely reported in the literature.From this, it follows that the conformation brought about by addition of ATP (or ADP.AlFx) can be assigned as a post-hydrolysis state (consistent with the standard interpretation of ADP.AlFx), because it maps neither to the pre-hydrolysis ATP nor ADP bound states.
The second set of comments by referee 3 focus on the PrlA4 variant.They make several assertions, which reflect a miss-understanding of the mechanism of protein transport, which are better addressed point-by-point: "Concerning the explanation that Τopen of Prl4 dictates the translocation process, do the authors think that a single opening event (e.g., within 4 ms) could allow the passage of an entire protein strand?"As we state in the manuscript, transport is rate limited by passage of bulky and positively charged regions of pre-protein.It is the transport of these regions only (not the entire polypeptide) that require the channel to be open for ~4 ms.

"If not, when a protein peptide is present in the channel without signal sequence peptides (SS), will the FRET signal indicate an open state or a closed state?
In other words, do the FRET experiments in this report distinguish between the opening of the lateral gate induced by SS (at the initialization stage, fig.4a), SS and a translocating peptide inside the channel (at the early translocation stage), and a peptide alone in the channel (at the middle and later translocation stages)?The authors do not seem to have considered this difference.However, in the reviewer's opinion, it is crucial to define the relationship between these various "open" states and the FRET signal before proceeding with further analysis."Because the signal sequence inserts as a hairpin with the early mature domain of the pre-protein and remains associated with the channel throughout the entire transport process, these are not in fact distinct states.

"Presumably, an idle channel has a closed lateral gate (LG), an active channel with SS and a translocating peptide has an open LG. An active channel with only a translocating peptide has an
LG opening somewhere between these two states.Neglecting to address this issue could potentially affect the interpretation of the Τopen/Τclosed plots for the ATP+proSpy sample." No presumption here is necessary, our data show that this is not the case.The channel is in dynamic equilibrium between two distinct states (open and closed), regardless of nucleotide or pre-protein occupancy.This is the crux of the paper.
We look forward to working with you towards a resolution to the differences between ourselves (and reviewers 1 and 2), to those of reviewer 3. Congratulations!I'll be really happy to see this work in our pages.
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Fig 5d .
Fig 5d.It would be preferable to label in this figure which is the variant data and which the wild type rather than relying on carrying over colours from other panels.What are the grey dotted lines?Are they necessary?Should they be arrows?Fig 5e.What do the comments written on the panel mean?Topen is presumably from the data shown but is the efficiency of translocation is from elsewhere and so can't be calculated from this graph?This should be made clear.R1-A8: We have added labels to panels Fig 5D and E as suggested.We also opted to remove the grey dashed lines from Fig 5D (originally added to help guide the reader) to avoid any confusion.We have added in parentheses to the annotations in Fig 5E to inform the reader where the data comes from.
3. Since the authors have determined the rate constants in the conformational transitions of the Sec translocon, it should be possible to calculate the energy of each state and include those values in the free energy profiles.R2-A3: We thank Reviewer #3 for the suggestion and we have now updated Fig 6A to reflect the changes suggested (see response R1-A9 above).
2. ADP.AlFx and ADP.BeFx are non-hydrolysable and exhibit a high affinity for SecA-SecY.The addition of these compounds to SecA-SecY can stabilize the complex in an open conformation, as demonstrated in the structure by Zimmer J et al.Nature, 2008.The observed increase in open time in the FRET experiments could be attributed to the open conformation of SecY in the structure.3. It is not surprising to observe similar LG dynamics in SecA-SecY between ATP binding and ADP.AlFx binding.Rather than proposing a new PH state as suggested by the authors, a more straightforward interpretation could be that ATP binding also keeps the channel open due to the structural similarity between ATP and ADP.AlFx.4. The AMPPNP data presented in the revised manuscript introduce further confusion.As cited by the authors, Fak JJ et al.
Your sincerely, Ian Collinson on behalf of the author team 6th Nov 2023 3rd Authors' Response to ReviewersAll editorial and formatting issues were resolved by the authors.to inform you that your manuscript has been accepted for publication in The EMBO Journal.
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